Spatially Resolved Force Spectroscopy
The microbial surface is rarely of homogeneous composition, but is rather made of a complex mixture of macro-molecules. As a result, a single cell can show important lateral variations of physical properties. Until recently, such local variations of properties were very difficult to measure, especially on hydrated, living cells. With spatially resolved force spectroscopy, it is possible to map the distribution of parameters such as adhesion and elasticity at the subcellular level.[25] The power of this approach is illustrated in Fig. 5. During cell growth, disruption of cell wall layers may occur and newly formed material may accumulate in localized regions, leading to heterogeneous surface properties. For germinating spores of Phanero-chaete chrysosporium,[26] the heterogeneous surface morphology (Fig. 5A) was directly correlated with differences in adhesion forces (Fig. 5B). The strong adhesion forces measured on localized zones were suggested to be responsible for cell aggregation observed during germination.
Another example of surface physical heterogeneity is found in yeast during budding. While chitin is usually not found in the cell walls of S. cerevisiae, it accumulates in the very localized region of the wall involved in budding where it may act as a stiffening agent. In this context, force-volume images were recently recorded in parallel with topographic images on the surface of budding yeast cells.[27] In Fig. 5C,D, it can be seen that the force map was highly contrasted, the bud scar region being clearly darker compared to the surrounding cell surface. These data indicated that the bud scar was qualitatively stiffer than the surrounding cell wall, a finding that was further supported by theoretical treatment of the force curves (see next section).
Fig. 5 Mapping adhesion forces and cell wall elasticity using spatially resolved force spectroscopy. (A) Deflection image (2 x 2 mm) and (B) adhesion map (z range = 10 nN) acquired on the same area of a germinating spore of P. chrysosporium; the adhesion map was obtained by recording 64 x 64 force-distance curves, calculating the adhesion force for each force curve, and displaying adhesion force values as grey levels (brighter contrast means larger adhesion forces). (C) Deflection image (1.5 x 1.5 mm) and (D) force map obtained for a S. cerevisiae cell after cell division; the force map, qualitatively reflecting the sample mechanical properties, was generated by recording 32x 32 force-distance curves and taking a slice of the force volume at a given sample height in the contact region of the curves (brighter contrast means softer properties).
Elasticity of Cell Walls
The mechanical properties of microbial layers can be addressed on a local scale using AFM force measurements. A first approach consists in pressing isolated cell wall material into a groove in a solid surface with the AFM tip and then deducing the elastic modulus from the applied force vs. depression distance curve. In a pioneering study, this ”depression technique” was used to measure the elastic modulus of the sheath of the archeon Methanospirillum hungatei GP1.[28] The large modulus values (20-40 GPa) indicated that this single-layered structure of unusual strength could withstand an internal pressure of 400 atm. For murein sacculi of Gram-negative bacteria in the hydrated state, elastic moduli of 25 MPa were measured, which are in excellent agreement with theoretical calculation of the elasticity of the peptidogly- can network.[29]
For whole cells, quantitative information on sample elasticity is obtained by converting force curves into force vs. indentation curves and analyzing them with theoretical models.[30] Using this ”nanoindentation method,” the wall compressibility of whole Magnetospirillum gryphis-waldense cells was determined to be about 42 mN/m.[31] Interestingly, local nanoindentation measurements on S. cerevisiae revealed significant variation of elasticity across the cell surface: Young’s modulus was 6 MPa on the bud scar and 0.6 MPa on the surrounding cell surface.1-27-1 These measurements provided the first direct evidence that the chitinous bud scar in yeast is about 10 times stiffer than the surrounding nonchitinous cell wall.
Chemically Functionalized Tips
Because commercial AFM tips usually have a poorly defined surface chemistry, they are not suited for quantitative surface force measurements. Tips of well-defined chemical composition can be created by function-alizing the surface with organic monolayers (alkanethiols or silanes) terminated by specific functional groups.[32] In doing so, researchers can measure local surface forces and physicochemical properties such as surface charge and surface hydrophobicity. Ahimou et al.[33] used AFM tips functionalized with ionizable carboxyl groups (COO-/ COOH) to probe the surface charges of S. cerevisiae at the nanometer level (Fig. 6). Force-distance curves were strongly influenced by pH: While no adhesion was measured at neutral/alkaline pH, multiple adhesion forces were recorded at acidic pH. Adhesion maps always showed fairly homogeneous contrast, indicating that the cell surface properties were homogeneous. The change of adhesion forces as a function of pH was interpreted as resulting from a change of cell surface electrostatic properties, a claim that was supported by the correlation obtained between the AFM titration curve constructed by plotting the adhesion force vs. pH and the electrophoretic mobility vs. pH curve obtained by classical microelectro-phoresis. Using a similar approach, it has been shown that hydrophilic (OH) and hydrophobic (CH3) tips can be used to map cell surface hydrophobicity.[34] These studies demonstrate that chemically functionalized tips enable quantitative measurement of surface properties at the subcellular level.
Fig. 6 Use of chemically functionalized tips for mapping cell surface charges. The left panel presents force-distance curves recorded in solutions of varying pH between the surface of S. cerevisiae and an AFM tip functionalized with carboxyl groups. The right panel shows adhesion force maps recorded at pH 7 and pH 4. The differences in adhesion forces observed with pH were related to a change of ionization state of the cell surface.
Biologically Functionalized Tips
Atomic force microscopy tips can also be functiona-lized with biomolecules to measure receptor-ligand interactions. A typical experiment consists in attaching ligands to the AFM tip and receptors to a solid substrate, or vice versa, bringing the modified surfaces in contact, and then measuring the unbinding force by pulling the tip away from the substrate. Successful measurements require that the binding of the biomole-cules to the tip and substrate is much stronger than the intermolecular force. This is typically achieved by using the chemisorption of alkanethiols on gold or the covalent binding of silanes on silicon oxide. Using this technique, a variety of intermolecular forces have been measured in recent years, among which the forces between biotin-avidin, antibody-antigen, complementary strands of DNA, carbohydrate-carbohydrate, and lectin-carbohydrate.[16,35,36]
Although they are not yet widely used in microbiology, AFM tips modified with biomolecules have a promising potential to measure receptor-ligand interaction forces associated with cell surfaces. This approach was recently applied to measure the specific interactions involved in yeast flocculation, an aggregation event of crucial importance in fermentation technology.[37] The yeast Saccharomyces carlsbergensis is known to flocculate according to a lectin-mediated mechanism in which active lectin receptors appear at the cell surface in the stationary phase and bind mannose residues of adjacent cells (Fig. 7). Atomic force microscopy tips functionalized with oligo-glucose carbohydrates were used to record force-distance curves on yeast cells. While nonflocculating exponential cells showed no or poor adhesion, stationary cells showed adhesion forces of 121 pN (Fig. 7). Control experiments (blocking with mannose, use of a nonflocculating strain) lead to the conclusion that the measured adhesion forces reflected individual lectin-carbohydrate interactions involved in yeast flocculation. This type of experiments should have an important impact on medicine because they may be used to measure the interaction forces between pathogens and host cells.
Fig. 7 Use of biologically functionalized tips for probing cell surface receptors. Optical micrographs of S. carlsbergensis cells in the exponential phase (left) and in the stationary phase (right). Force curves recorded between exponential (left) or stationary (right) cells and AFM tips functionalized with oligoglucose carbohydrates. The adhesion force measured on stationary cells was attributed to the specific interaction between cell surface lectins and glucose residues on the AFM tip.
”Cell probes” represent another type of biologically modified tips that are useful for measuring interaction forces between cells and solid substrata. Here a crucial issue is to ensure that both the metabolic activity and natural surface architecture are preserved during immobilization procedures. In this context, Bowen and co-workers developed an approach in which a single living cell is glued on the apex of a tipless cantilever using a micromanipulator. In doing so, the adhesion between fungal spores,[38] yeast cells,[39] and various solid surfaces was directly quantified. Another nondestructive strategy consists in coating an amino-functionalized bead with living bacterial cells and then attaching it to an AFM cantilever. This approach enabled the forces between Shewanella oneidensis bacteria and mineral surfaces to be quantitatively measured;[40] an important finding was that stronger adhesion energies were measured under anaerobic conditions. Accordingly, AFM-based force spectros-copy measurements provide a means to study a variety of cellular interactions at the molecular level.
Mechanical Properties of Single Molecules
Remarkably, AFM can also be used to probe intramolecular forces associated with individual molecules, thereby providing quantitative information on the elasticity of the molecules, on conformational transitions along the chains, on the mechanical stability of chemical bonds, and on secondary structures. In recent years, these single-molecule force spectroscopy experiments have been applied to a variety of biomolecules, including DNA, proteins, and polysaccharides.[16,35,36,41-43] Outstanding work has also been carried out on isolated microbial cell surface layers. In the first such study, Muller et al.[44] combined AFM imaging and force spectroscopy to unzip proteins from the hexagonally packed intermediate (HPI) layer of Deino-coccus radiodurans. Force-extension curves recorded for the inner surface of the HPI layer showed saw-tooth patterns with six force peaks of about 300 pN. This behavior was attributed to the sequential pulling out of the protomers of the hexameric HPI protein complex. After recording the force curve, a molecular defect the size of a hexameric complex was clearly visualized by means of high-resolution imaging. In another study, Oesterhelt et al.[45] unraveled the unfolding pathways of individual bacteriorhodopsins. Molecules were individually extracted from the membrane, anchoring forces of 100200 pN being found for the different helices. Upon retraction, the helices were found to unfold and the force spectra revealed the individuality of the unfolding pathways. Scheuring et al.[46] combined AFM imaging and single-molecule force spectroscopy to gain insight into the mechanical properties of the Coryne-bacterium glutamicum S-layer. The results provided a basis for understanding the extraordinary stability of these protein networks that protect microorganisms from hostile environment. The above studies demonstrate that AFM imaging and force spectroscopy is a powerful approach to address the structure-function relationships of microbial cell surface layers at the single molecule level.
Single-molecule elasticity experiments on living cells are very challenging in view of the complex and dynamic nature of their surface architecture. Recent studies indicate that progress is being made in this area. Atomic force microscopy was used to pull on macromolecules exposed at the surface of living A. oryzae spores with the aim to gain insight into their elasticity.[22] As mentioned earlier, topographic imaging revealed that, upon germination, the crystalline-like surface of dormant spores changed into a layer of soft granular material attributed to cell surface polysaccharides. This structural modification was correlated with a change of molecular interactions: While dormant spores showed poor adhesion, germinating spores showed attractive forces of 400 pN magnitude along with elongation forces that were attributed to the stretching of the cell surface polysaccharides (Fig. 8). This interpretation was supported by the finding that fitting the curves with a theoretical model from statistical mechanics yielded parameters similar to those reported for individual amylose and dextran molecules. It was concluded that the stickiness and flexibility of cell surface polysaccharide chains may promote spore aggregation via macromolecular bridging interactions between opposing cells.
Single-molecule force spectroscopy has also been applied to bacterial cells. The elasticity and adhesion of surface polymers from Pseudomonas putida was measured in solvents spanning a range of polarity and ionic strengths.[47] Adhesion forces in water and formamide were about the same and smaller than forces observed in methanol. Different models from statistical mechanics were used to analyze the force-extension profiles. Although biopolymer contour lengths varied over a wide range in all solvents, shorter lengths were observed when salt was present, indicating that the polymer chains were less extended in the presence of salt. In a related study, the heterogeneity in bacterial surface macromol-ecules was probed on the surface of P. putida.[48] Force measurements on different bacterial cells showed a range of adhesion affinities and polymer lengths, but substantial heterogeneity was also observed in the curves on a single bacterium. These experiments indicated that heterogeneity in biopolymer properties on an individual bacterium and within a population of bacterial cells may be much greater than previously believed and should be incorporated into models of bacterial adhesion. Summarizing, the above studies indicate that single-molecule force measurements should be very useful in future microbiological research for elucidating the properties of cell surface macromolecules.
Fig. 8 Pulling on cell surface macromolecules. Force-distance curve recorded under water between a silicon nitride tip and the surface of a germinating A. oryzae spore (thin line). The elongation force was well described by an extended freely jointed chain model (thick line) with parameters consistent with the stretching of a single polysaccharide chain.
CONCLUSION
Atomic force microscopy imaging and force spectroscopy have recently opened a wide spectrum of novel applications for microbiologists and biophysicists. Using imaging in aqueous solution, microscopists can visualize cell surface nanostructures (surface layers, appendages), follow physiological changes (germination, growth), and monitor the effect of external agents (antibiotics, metals) in real time. By recording force-distance curves on cell surfaces, researchers can learn about local biomolecular interactions and physical properties. Spatially resolved force mapping offers a means to determine physical/ chemical heterogeneities at the subcellular level, thereby providing complementary information to classical characterization methods. Force spectroscopy can be used to quantitatively probe the elasticity of cell wall components and whole cells. Functionalizing the AFM tip with chemical groups, biomolecules, and living cells enables quantitative measurements of surface charge, surface hydrophobicity, receptor-ligand interactions, and cell- material interactions. Finally, single-molecule force spec-troscopy can be applied to cell surface molecules to gain insight into their mechanical properties. Clearly, these AFM-based experiments contribute to improve our understanding of the structure-function relationships of microbial cell surfaces and will have considerable impact on biotechnology and medicine.
